亚洲玉米螟Bt抗性种群的遗传多样性分析
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摘要
近20年来,在北美、西欧、南美、菲律宾等20多个国家大面积商业化应用Bt玉米防治欧洲玉米螟Ostrinia nubilalis(Hübner)等鳞翅目害虫的实践证明,其不可避免地胁迫靶标害虫产生遗传分化,出现新的抗性种群。研究精准有效的抗性检测方法,将为抗性监测和抗性治理策略的有效性提供科学依据。本研究基于高通量测序获得的亚洲玉米螟Ostrinia furnacalis(Guenée)转录组数据,应用MISA (MicroSAtellite)软件搜索SSR位点,从61 622条EST(Expressed Sequence Tag)序列中获得了3 467个SSR位点。通过设计、筛选,共获得3 316对特异性引物,从中挑选了150对引物进行PCR扩增,共有51对扩增出目的条带,对亚洲玉米螟敏感种群(ACB-BtS)及5个Bt毒素抗性种群(ACB-AbR、ACB-AcR、ACB-AhR、ACB-FR、ACB-IeR)进行多态性检测,最终得到20条高多态性引物。利用这20对微卫星引物共检测到126个等位基因,平均每个位点6.3个。不同Bt抗性种群间产生了一定程度的遗传分化,种群间的平均遗传分化系数(F_(st))为0.195 9,即说明种群间的遗传变异为19.6%。根据遗传距离建立了UPGMA系统发育树,显示6个种群的相似度,即ACB-AbR与ACB-AcR相似度高。种群变异相似度规律与已报道的亚洲玉米螟对各Bt毒素的交互抗性规律相一致。本研究发现的SSR位点可作为亚洲玉米螟不同Bt毒素抗性种群的分子检测方法。
In the past two decades, the commercialization of Bt corn for protecting corn against lepidopteran pests such as the European corn borer, Ostrinia nubilalis(Hübner), has been widely used in more than 20 countries in North America, Western Europe, South America, and the Philippines. Accordingly, evolution of resistance has been reported in target pests driven by widespread Bt corn. Accurate and effective resistance testing methods will provide scientific basis for the effectiveness of resistance monitoring and resistance management strategies. Using MISA(MicroSAtellite) software to analyze 61 622 ETS sequences of the transcriptome database of the Asian corn borer, O.furnacalis(Guenée) and 3 467 SSR loci were identified. By designing and screening, we designed 3 316 pairs of specific primers, of which 150 pairs of primers were selected for PCR amplification, and there were 51 pairs producing amplification bands of expected sizes. Through polymorphism detection, 20 highly polymorphic primers were obtained among susceptible Asian corn borer populations and 5 Bt-resistant populations(ACB-AbR, ACB-AcR, ACB-AhR, ACB-FR and ACB-IeR). By targeting these 20 highly polymorphic loci to analyze the genetic diversities of different Bt-resistant populations of the Asian corn borer, 126 alleles were detected by using those 20 pairs of microsatellite primers, with an average of 6.3 alleles per locus. The average genetic differentiation coefficient(F_(st)) among populations was 0.195 9 in the Asian corn borer, indicating that 19.6% genetic differentiation existed among the populations. UPGMA phylogenetic tree was established based on genetic distances, showing similarity among the six populations, i.e., high similarity between ACB-AbR and ACB-AcR, and high similarity between ACB-FR and ACB-IeR.The pattern of genetic similarity among the strains was similar to the previously reported pattern of cross-resistance to Bt toxins. Those 20 pairs of microsatellite primers could be used to establish the molecular detection method for monitoring resistance to Bt toxins in Asian corn borer.
In the past two decades, the commercialization of Bt corn for protecting corn against lepidopteran pests such as the European corn borer, Ostrinia nubilalis(Hübner), has been widely used in more than 20 countries in North America, Western Europe, South America, and the Philippines. Accordingly, evolution of resistance has been reported in target pests driven by widespread Bt corn. Accurate and effective resistance testing methods will provide scientific basis for the effectiveness of resistance monitoring and resistance management strategies. Using MISA(MicroSAtellite) software to analyze 61 622 ETS sequences of the transcriptome database of the Asian corn borer, O.furnacalis(Guenée) and 3 467 SSR loci were identified. By designing and screening, we designed 3 316 pairs of specific primers, of which 150 pairs of primers were selected for PCR amplification, and there were 51 pairs producing amplification bands of expected sizes. Through polymorphism detection, 20 highly polymorphic primers were obtained among susceptible Asian corn borer populations and 5 Bt-resistant populations(ACB-AbR, ACB-AcR, ACB-AhR, ACB-FR and ACB-IeR). By targeting these 20 highly polymorphic loci to analyze the genetic diversities of different Bt-resistant populations of the Asian corn borer, 126 alleles were detected by using those 20 pairs of microsatellite primers, with an average of 6.3 alleles per locus. The average genetic differentiation coefficient(F_(st)) among populations was 0.195 9 in the Asian corn borer, indicating that 19.6% genetic differentiation existed among the populations. UPGMA phylogenetic tree was established based on genetic distances, showing similarity among the six populations, i.e., high similarity between ACB-AbR and ACB-AcR, and high similarity between ACB-FR and ACB-IeR.The pattern of genetic similarity among the strains was similar to the previously reported pattern of cross-resistance to Bt toxins. Those 20 pairs of microsatellite primers could be used to establish the molecular detection method for monitoring resistance to Bt toxins in Asian corn borer.
引文
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[2] MCGAUGHEY W H, BEEMAN R W. Resistance to Bacillus thuringiensis in colonies of indianmeal moth and almond moth (Lepidoptera: Pyralidae) [J]. Journal of Economic Entomology, 1988, 81(1): 28-33.
[3] MüLLERCOHN J, CHAUFAUX J, BUISSON C, et al. Spodoptera littoralis (Lepidoptera: Noctuidae) resistance to CryIC and cross-resistance to other Bacillus thuringiensis crystal toxins [J]. Journal of Economic Entomology, 1996, 89(4): 791-797.
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[7] LEE M K, RAJAMOHAN F, GOULD F, et al. Resistance to Bacillus thuringiensis CryIA delta-endotoxins in a laboratory-selected Heliothis virescens strain is related to receptor alteration [J].Applied & Environmental Microbiology,1995,61(11): 3836-3842.
[8] STONE T B, SIMS S R, MARRONE P G. Selection of tobacco budworm for resistance to a genetically engineered Pseudomonas fluorescens, containing the δ-endotoxin of Bacillus thuringiensis subsp. kurstaki [J]. Journal of Invertebrate Pathology, 1989, 53(2): 228-234.
[9] 梁革梅, 谭维嘉. 棉铃虫Bt抗性种群的RAPD—PCR初步分析[J]. 植物保护, 2000, 26(3): 4-6.
[10] 雷仲仁, 郭予元, 李莉, 等. 棉铃虫抗药性种群的RAPD分析[C]//植物保护21世纪展望暨全国青年植物保护科技工作者学术研讨会, 1998:548-550.
[11] 潘志萍, 曾玲, 温硕洋. 桔小实蝇抗性品系的微卫星DNA分析[J]. 昆虫学报, 2006, 49(5): 874-877.
[12] ZHANG Tiantao, HE Mingxia, GATEHOUSE A M R, et al. Inheritance patterns, dominance and cross-resistance of Cry1Ab-and Cry1Ac-selected (Guenée)[J]. Bautechnik, 2014, 74(6): 395-400.
[13] WANG Yueqin, YANG Jing, QUAN Yudong, et al. Characterization of Asian corn borer resistance to Bt toxin Cry1Ie [J]. Toxins, 2017, 9(6): 186.
[14] WANG Yueqin, WANG Yidong, WANG Zhenying, et al. Genetic basis of Cry1F-resistance in a laboratory selected Asian corn borer strain and its cross-resistance to other Bacillus thuringiensis toxins[J/OL]. PLoS ONE, 2016, 11(8): e0161189.
[15] SHABBIR M Z, QUAN Yudong, WANG Zhenying, et al. Characterization of the Cry1Ah resistance in Asian corn borer and its cross-resistance to other Bacillus thuringiensis toxins [J]. Scientific Reports, 2018, 8(1): 234.
[16] 贺明霞, 何康来, 王振营,等. Cry1Ie毒素胁迫下亚洲玉米螟的抗性发展及汰选种群对其他Bt毒素的交互抗性[J]. 昆虫学报, 2013, 56(10):1135-1142.
[17] NEI M. Genetic distance between populations [J]. American Naturalist, 1972, 106(949): 283-292.
[18] BOTSTEIN D, WHITE R L, SKOLNICK M, et al. Construction of a genetic linkage map in man using restriction fragment length polymorphisms [J]. American Journal of Human Genetics, 1980, 32(3): 314-331.
[19] 李伟丰, 杨朗, 唐侃, 等. 中国桔小实蝇种群的微卫星多态性分析[J]. 昆虫学报, 2007, 50(12): 1255-1262.
[20] BALLOUX F, LUGON-MOULIN N. The estimation of population differentiation with microsatellite markers [J]. Molecular Ecology, 2002, 11(2): 155-165.
[21] CHAMBERS J A, JELEN A, GILBERT M P, et al. Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai [J]. Journal of Bacteriology, 1991, 173(13): 3966-3976.
[22] H?FTE H, WHITELEY H R.Insecticidal crystal proteins of Bacillus thuringiensis [J]. Microbiological Reviews, 1989, 53(2): 242-255.
[23] DENOLF P, JANSENS S, PEFEROEN M, et al. Two different Bacillus thuringiensis delta-endotoxin receptors in the midgut brush border membrane of the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae)[J]. Applied & Environmental Microbiology, 1993, 59(6): 1828-1837.
[24] WOLFERSBERGER M G. The toxicity of two Bacillus thuringiensis δ-endotoxins to gypsy moth larvae is inversely related to the affinity of binding sites on midgut brush border membranes for the toxins [J].Experientia,1990,46(5):475-477.
[25] XU Lina, WANG Zhenying, ZHANG Jie, et al. Cross-resistance of Cry1Ab-selected Asian corn borer to other cry toxins[J].Journal of Applied Entomology,2010,134(5):429-438.
[26] JIANG Fan, ZHANG Tiantao, BAI Shuxiong, et al. Evaluation of Bt corn with pyramided genes on efficacy and insect resistance management for the Asian corn borer in China [J/OL]. PLoS ONE, 2016, 11(12): e0168442.
[2] MCGAUGHEY W H, BEEMAN R W. Resistance to Bacillus thuringiensis in colonies of indianmeal moth and almond moth (Lepidoptera: Pyralidae) [J]. Journal of Economic Entomology, 1988, 81(1): 28-33.
[3] MüLLERCOHN J, CHAUFAUX J, BUISSON C, et al. Spodoptera littoralis (Lepidoptera: Noctuidae) resistance to CryIC and cross-resistance to other Bacillus thuringiensis crystal toxins [J]. Journal of Economic Entomology, 1996, 89(4): 791-797.
[4] ESTADA U, FERRE J. Binding of insecticidal crystal proteins of Bacillus thuringiensis to the midgut brush border of the cabbage looper, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), and selection for resistance to one of the crystal proteins[J]. Applied & Environmental Microbiology, 1994, 60(10): 3840-3846.
[5] MOAR W J, PUSZTAICAREY M, FAASSEN H V, et al. Development of Bacillus thuringiensis CryIC resistance by Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae)[J]. Applied & Environmental Microbiology,1995,61(6):2086-2092.
[6] GOULD F, MARTINEZ-RAMIREZ A, ANDERSON A, et al. Broad-spectrum resistance to Bacillus thuringiensis toxins in Heliothis virescens [J]. Proceedings of the National Academy of Sciences of the United States of America, 1992, 89(17): 7986-7990.
[7] LEE M K, RAJAMOHAN F, GOULD F, et al. Resistance to Bacillus thuringiensis CryIA delta-endotoxins in a laboratory-selected Heliothis virescens strain is related to receptor alteration [J].Applied & Environmental Microbiology,1995,61(11): 3836-3842.
[8] STONE T B, SIMS S R, MARRONE P G. Selection of tobacco budworm for resistance to a genetically engineered Pseudomonas fluorescens, containing the δ-endotoxin of Bacillus thuringiensis subsp. kurstaki [J]. Journal of Invertebrate Pathology, 1989, 53(2): 228-234.
[9] 梁革梅, 谭维嘉. 棉铃虫Bt抗性种群的RAPD—PCR初步分析[J]. 植物保护, 2000, 26(3): 4-6.
[10] 雷仲仁, 郭予元, 李莉, 等. 棉铃虫抗药性种群的RAPD分析[C]//植物保护21世纪展望暨全国青年植物保护科技工作者学术研讨会, 1998:548-550.
[11] 潘志萍, 曾玲, 温硕洋. 桔小实蝇抗性品系的微卫星DNA分析[J]. 昆虫学报, 2006, 49(5): 874-877.
[12] ZHANG Tiantao, HE Mingxia, GATEHOUSE A M R, et al. Inheritance patterns, dominance and cross-resistance of Cry1Ab-and Cry1Ac-selected (Guenée)[J]. Bautechnik, 2014, 74(6): 395-400.
[13] WANG Yueqin, YANG Jing, QUAN Yudong, et al. Characterization of Asian corn borer resistance to Bt toxin Cry1Ie [J]. Toxins, 2017, 9(6): 186.
[14] WANG Yueqin, WANG Yidong, WANG Zhenying, et al. Genetic basis of Cry1F-resistance in a laboratory selected Asian corn borer strain and its cross-resistance to other Bacillus thuringiensis toxins[J/OL]. PLoS ONE, 2016, 11(8): e0161189.
[15] SHABBIR M Z, QUAN Yudong, WANG Zhenying, et al. Characterization of the Cry1Ah resistance in Asian corn borer and its cross-resistance to other Bacillus thuringiensis toxins [J]. Scientific Reports, 2018, 8(1): 234.
[16] 贺明霞, 何康来, 王振营,等. Cry1Ie毒素胁迫下亚洲玉米螟的抗性发展及汰选种群对其他Bt毒素的交互抗性[J]. 昆虫学报, 2013, 56(10):1135-1142.
[17] NEI M. Genetic distance between populations [J]. American Naturalist, 1972, 106(949): 283-292.
[18] BOTSTEIN D, WHITE R L, SKOLNICK M, et al. Construction of a genetic linkage map in man using restriction fragment length polymorphisms [J]. American Journal of Human Genetics, 1980, 32(3): 314-331.
[19] 李伟丰, 杨朗, 唐侃, 等. 中国桔小实蝇种群的微卫星多态性分析[J]. 昆虫学报, 2007, 50(12): 1255-1262.
[20] BALLOUX F, LUGON-MOULIN N. The estimation of population differentiation with microsatellite markers [J]. Molecular Ecology, 2002, 11(2): 155-165.
[21] CHAMBERS J A, JELEN A, GILBERT M P, et al. Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai [J]. Journal of Bacteriology, 1991, 173(13): 3966-3976.
[22] H?FTE H, WHITELEY H R.Insecticidal crystal proteins of Bacillus thuringiensis [J]. Microbiological Reviews, 1989, 53(2): 242-255.
[23] DENOLF P, JANSENS S, PEFEROEN M, et al. Two different Bacillus thuringiensis delta-endotoxin receptors in the midgut brush border membrane of the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae)[J]. Applied & Environmental Microbiology, 1993, 59(6): 1828-1837.
[24] WOLFERSBERGER M G. The toxicity of two Bacillus thuringiensis δ-endotoxins to gypsy moth larvae is inversely related to the affinity of binding sites on midgut brush border membranes for the toxins [J].Experientia,1990,46(5):475-477.
[25] XU Lina, WANG Zhenying, ZHANG Jie, et al. Cross-resistance of Cry1Ab-selected Asian corn borer to other cry toxins[J].Journal of Applied Entomology,2010,134(5):429-438.
[26] JIANG Fan, ZHANG Tiantao, BAI Shuxiong, et al. Evaluation of Bt corn with pyramided genes on efficacy and insect resistance management for the Asian corn borer in China [J/OL]. PLoS ONE, 2016, 11(12): e0168442.